Detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study.

AIM: We have designed a study with the objective of describing the clinical impact of other viruses different from the respiratory syncytial virus (RSV) in hospitalized infants with bronchiolitis. METHODS: A 3 year prospective study was conducted on infants admitted to the Paediatrics Department of the Severo Ochoa Hospital (Spain). We studied the frequency of 16 respiratory viruses. Clinical characteristics of RSV-only infections were compared with other single agent viral infections. RESULTS: Positive results were confirmed in 275 (86.5%) of the 318 children studied. A single virus was detected in 196 patients and 79 were dual or multiple viral infections. RSV was detected in 61.3% of total bronchiolitis. Rhinovirus (RV) was 17.4% of the identified virus, followed by human bocavirus (HBoV), adenovirus and metapneumovirus (hMPV). Only RV, HBoV and hMPV were significant as single infections. RSV patients were younger than HBoV (p > 0.0001) and hMPV (p = 0.025). Seasonality was clearly different between them. Children with RSV infection needed treatment in the intensive care unit more frequently than others. CONCLUSIONS: In hospitalized infants, RSV was the most frequent agent in bronchiolitis in winter, but other viruses were present in 47% of the patients. RV, HBoV and hMPV had a significant proportion of single infections. Clinical characteristics were similar amongst them, but seasonality was clearly different.

Human case of swine influenza A (H1N1), Aragon, Spain, November 2008.

A human case of swine influenza A (H1N1) in a 50-year-old woman from a village near Teruel (Aragon, in the north-east of Spain), with a population of about 200 inhabitants, has been reported in November 2008.

Human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5.

BACKGROUND: Human metapneumovirus (hMPV) infection plays an important role in the pediatric respiratory infections. However, little is known about the relationship between hMPV-bronchiolitis and the development of subsequent wheezing. The aim of this study was to evaluate the outcome at third to fifth year after admission for hMPV-bronchiolitis and to compare it with children admitted for respiratory syncytial virus (RSV)-bronchiolitis and with children without lower respiratory disease in the first 2 years of life. METHODS: We studied 55 children (23 hMPV and 32 RSV acute bronchiolitis) aged 3-5 years and hospitalized during the seasons 2000-2005. Thirty-eight children admitted due to acute rotavirus gastroenteritis in the same seasons, and without bronchiolitis during infancy, were also analyzed. Clinical data were collected through structured clinical interviews at the follow-up visit. Skin prick tests to food and inhaled allergens were performed. RESULTS: Asthma was significantly more frequent in children with hMPV-bronchiolitis (odds ratio (OR) = 5.21), RSV-bronchiolitis (OR = 4.68), allergic rhinitis (OR = 1.7), and siblings with atopy (OR = 1.75). In the multivariate analyses, hMPV was the most important risk factor for asthma in preschool years (OR = 15.9), followed by RSV-bronchiolitis (OR = 10.1) and allergic rhinitis (OR = 4.9). No differences were found with respect to allergic rhinitis, atopic dermatitis, food allergy, proportion of positive prick tests, family history of atopy, and asthma. CONCLUSIONS: hMPV-bronchiolitis in infancy was strongly associated with asthma as well as other forms of bronchial obstructive disease at third and fifth year of life. This association is, at least, as strong as the association observed with RSV infections.

Understanding the transmission dynamics of respiratory syncytial virus using multiple time series and nested models.

The nature and role of re-infection and partial immunity are likely to be important determinants of the transmission dynamics of human respiratory syncytial virus (hRSV). We propose a single model structure that captures four possible host responses to infection and subsequent reinfection: partial susceptibility, altered infection duration, reduced infectiousness and temporary immunity (which might be partial). The magnitude of these responses is determined by four homotopy parameters, and by setting some of these parameters to extreme values we generate a set of eight nested, deterministic transmission models. In order to investigate hRSV transmission dynamics, we applied these models to incidence data from eight international locations. Seasonality is included as cyclic variation in transmission. Parameters associated with the natural history of the infection were assumed to be independent of geographic location, while others, such as those associated with seasonality, were assumed location specific. Models incorporating either of the two extreme assumptions for immunity (none or solid and lifelong) were unable to reproduce the observed dynamics. Model fits with either waning or partial immunity to disease or both were visually comparable. The best fitting structure was a lifelong partial immunity to both disease and infection. Observed patterns were reproduced by stochastic simulations using the parameter values estimated from the deterministic models.

[Human bocavirus infections in Spanish 0-14 year-old: clinical and epidemiological characteristics of an emerging respiratory virus]. / Infecciones por bocavirus humano en niños españoles: características clínicas y epidemiológicas de un virus respiratorio emergente.

INTRODUCTION: In 2005 a new respiratory virus, called human bocavirus (HBoV), was cloned from respiratory samples from Swedish infants and children with lower respiratory tract infections. OBJECTIVES: To determine whether HBoV has circulated in Spain, estimate the frequency of HBoV infections in patients hospitalized for respiratory infection and describe the clinical and epidemiological characteristics of these patients. PATIENTS AND METHODS: We performed a descriptive prospective study of confirmed HBoV infections in patients aged < 14 years old, hospitalized for respiratory infections between October 2004 and June 2005. Virologic diagnosis was based on multiple RT-PCR for respiratory syncytial virus (RSV) A and B, influenza A,B, and C, parainfluenza 1-4, adenovirus and rhinovirus; PCR was used for human metapneumovirus (hMPV) and PCR in nasopharyngeal aspirates was used for HBoV. The clinical and epidemiological characteristics of patients were analyzed. RESULTS: Fifty-two cases of HBoV infection were detected, representing 17.1% (95% CI: 13% a 21%) of patients hospitalized for respiratory infections. HBoV was the third most frequent viral agent after RSV (30%) and rhinovirus (25%). In 39 patients (71.1%) coinfection with another respiratory virus was detected. Fifty percent of the patients were aged less than 13.6 months and 75% were aged less than 2 years. The most frequent diagnoses were recurrent wheezing (55.8%), bronchiolitis (21.2%) and pneumonia (15.4%). Clinical sepsis with petechial exanthema was found in two patients. Fever > 38 degrees C was found in 72.1% and radiological infiltrate in 44%. Hypoxia was present in 55.8 % of the patients. HBoV was isolated in distinct episodes in two patients. Coinfections were similar to simple infections except that hypoxia was more frequent in the former (p = 0.038). CONCLUSIONS: HBoV is one of the most frequent viruses in severe respiratory infections in patients aged less than 14 years old. Only RSV and rhinovirus are more frequent. Coinfections are highly frequent. Most patients are infants with recurrent wheezing and bronchiolitis.

Prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in Spain.

Human metapneumovirus (hMPV), a condition recently described in the Netherlands, causes lower respiratory infections, particularly in young children and among the elderly. The objective of this study was to describe the characteristics of hMPV infections in hospitalized infants <2 years of age and to compare them to those of infections caused by respiratory syncytial virus (RSV). A prospective study was conducted on the clinical characteristics of infants admitted to hospital for respiratory infection through 5 years. Simultaneous detection of influenza A, B, and C viruses, RSV, and adenoviruses was performed in clinical samples by multiple reverse transcription nested-PCR assay. The presence of hMPV was tested in all samples using two separate RT-PCR tests. Some respiratory virus was detected in 70.5% of the 1,322 children included in the study. hMPV was found in 101 of the positive nasopharyngeal aspirates (10.8%), and was the most common virus after RSV and rhinovirus. Peak incidence was found in March. Over 80% of children were <12 months. The more common diagnoses were bronchiolitis (49.5%) and recurrent wheezing (45.5%). Fifty-four percent of cases required oxygen therapy and, one percent, assisted ventilation. Thirty percent were co-infections, with clinical characteristics indistinguishable from single infections. Seventy-one hMPV single infections were compared to 88 RSV single infections. hMPV infections were significantly more frequent than RSV in infants older than 6 months (P = 0.04). Recurrent wheezing was diagnosed more frequently in hMPV patients (P = 0.001). All other variables tested were similar, in both groups. hMPV was the third most frequent virus after RSV and rhinovirus in infants <2 years of age, hospitalized for respiratory infection, and was associated with bronchiolitis and recurrent wheezing. hMPV predominantly occurred in spring. Co-infections were frequent and clinically similar to single infections and RSV infections.

[Role of rhinovirus in respiratory tract infections in hospitalized children]. / Papel del rinovirus en las infecciones respiratorias en niños hospitalizados.

BACKGROUND: Rhinovirus is a recognized cause of common cold and has been shown to cause asthma exacerbations in adults and children. The burden of rhinovirus infections in hospitalized children has not been described in Spain. OBJECTIVE: To describe confirmed rhinovirus infections in children hospitalized for respiratory tract infection in a secondary public hospital. PATIENTS AND METHODS: We performed a prospective descriptive study in children admitted to hospital with fever or respiratory tract infection and with a positive isolation of rhinovirus in nasopharyngeal washings between September 2004 and July 2005. Virological diagnosis was made with direct immunofluorescent assay and/or reverse transcription-polymerase chain reaction in specimens obtained from nasopharyngeal washings. The clinical characteristics of the patients were analyzed. RESULTS: There were 76 children with rhinovirus infection, representing 25 % of admissions in 304 children with fever or respiratory tract infection. Rhinovirus was the second most frequent viral agent identified after respiratory syncytial virus (RSV) (29.9 % of admissions). Fifty-four children (71.1 %) were under 2 years of age. The most frequent clinical diagnoses were recurrent wheezing in 60.5 %, bronchiolitis in 23.7 %, pneumonia in 7.9 %, and upper respiratory tract infection in 5.3 %. Fever > 38 degrees C was present in 57.9 % of the patients and radiologic infiltrate was found in 23.7 %. Oxygen saturation less than 95 % was found in 43.4 % of the patients. Of 22 children aged more than 2 years, a diagnosis of asthmatic crisis was made in 21. CONCLUSIONS: Rhinoviruses were frequently identified in hospitalized children with respiratory tract disease and were the second most common viruses after RSV. In our series, it was the most frequent cause of recurrent wheezing in hospitalized children and the second most common cause in infants.

[Influenza virus infections in infants aged less than two years old]. / Infecciones por virus de la gripe en menores de dos años.

INTRODUCTION: Influenza is a major cause of respiratory tract illness in infants. The clinical characteristics of these infections are non-specific and the burden of influenza is frequently underestimated in very young children. The objective of this study was to describe confirmed influenza infections in infants < 2 years attended in a level II public hospital. A second-rate aim was to compare influenza infections in hospitalized infants with respiratory syncytial virus (VRS) infection in the same population. PATIENTS AND METHODS: We performed a descriptive, prospective study between 1991 and 2003 in infants younger than 24 months of age, who were admitted to the Severo Ochoa Hospital (Leganés. Madrid) with fever or respiratory tract infection. Virological diagnosis was made with direct immunofluorescent assay and/or reverse transcription-polymerase chain reaction on specimens obtained from nasopharyngeal washings. The patients' clinical characteristics were recorded. Patients with influenza infection were compared with a similar group of infants with RSV infection. RESULTS: We analyzed hospitalized 100 infants with influenza infection. Influenza caused 4.1% of the admissions in infants with fever or respiratory tract infection. Influenza A was isolated in 83%, influenza B in 12% and influenza C in 5% of the patients. The mean age of hospitalized infants was 8.3 +/- 5.9 months and the most frequent clinical diagnoses were bronchiolitis in 38%, recurrent wheezing in 25%, upper respiratory tract infection in 19% and pneumonia in 9%. Fever > 38 degrees C was present in 83% of the patients. Radiologic infiltrate was found in 65% of the children. Oxygen saturation less then 95% was present in 44%. In children under 6 months of age fever was less frequent (p = 0.049) and upper respiratory tract infection was more frequent (p = 0.01). Patients with influenza virus infection were older (p = 0.002), more frequently presented fever (p < 0.0001) and radiologic infiltrate (p < 0.001) than infants with RSV infection. Bronchiolitis was more frequent in the RSV group (p = 0.006). CONCLUSIONS: Influenza infection is a major cause of respiratory tract disease in hospitalized infants. It is an etiologic cause of bronchiolitis, recurrent wheezing, and fever and radiologic consolidations are frequent. Clinical presentation is milder in children under 6 month of age. The characteristics of influenza infection differ substantially from those of RSV infection.

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review.

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.

[A surveillance system for the study of the circulation of influenza in Madrid during the 1986-1991 quinquennium. The Influenza Surveillance Group of Madrid]. / Sistema de vigilancia para el estudio de la circulación gripal en Madrid durante el quinquenio 1986-1991. Grupo de Vigilancia de Gripe de Madrid.

BACKGROUND: The creation of a surveillance system allowing the study of the evolution of the tendency of influenza and act as an alert system for undertaking control measures is necessary due to the great variability characteristic of the circulation of influenza virus. The Madrid Influenza Surveillance Group was therefore designed. The activity carried out during its first five years of existence is evaluated. METHODS: The field work of the Group is based on the clinical study of patients with influenza, among which some are selected for virologic study with the aim to isolate and characterize the circulating virus. The data obtained by the work group itself are used as the dates of the System of Obligatory Disease Declaration and the System of Microbiologic Information as epidemiologic support to define the outbreaks observed. RESULTS: All the virus isolated were similar to those recommended by the World Health Organization for their inclusion in anti-influenza vaccination, with their circulation being largely reproduced throughout Europe. In three of the five seasons studied the type A virus prevailed leading to earlier outbreaks and a greater number of cases than in the two seasons with type B prevalence. CONCLUSIONS: The circulation of the influenza virus in Madrid coincides with that of the rest of Spain at both the maximum peak and relative incidence as well as the type of prevailing virus. The minor circulation detected by the Madrid Group is in concordance with the data provided by the other autonomic communities.

[Respiratory infections due to metapneumovirus in hospitalized infants]. / Infecciones respiratorias por metapneumovirus en lactantes hospitalizados.

BACKGROUND: Human metapneumovirus (hPMV) is a recently identified virus that is recognized as a cause of respiratory tract illness in the pediatric population. OBJECTIVES: To determine the incidence of respiratory tract infections caused by hPMV in hospitalized infants and to describe the clinical characteristics and possible presence of coinfection with other viral agents. PATIENTS AND METHODS: We performed a prospective study from September to June 2003 in all children aged less than 24 months who were admitted to the Severo Ochoa Hospital (Leganés, Madrid) with a respiratory tract infection. Virological diagnosis was made with a direct immunofluorescent assay and/or reverse transcriptase-polymerase chain reaction on specimens obtained from nasopharyngeal washing. Demographic and clinical data from patients with an hPMV respiratory tract infection were analyzed. RESULTS: During the study period, 200 infants were admitted with a respiratory tract infection, of which 18 (9 %) had an hPMV infection. HPMV was the viral agent isolated in 13.8 % of positive nasopharyngeal washings. All patients were admitted between March and April. The mean age was 6.7 +/- 6.1 months. The most common diagnoses were recurrent wheezing (55.5 %) and bronchiolitis (38.8 %). Oxygen therapy was required by 55.5 % of infants during hospitalization. Coinfection with other respiratory viruses was confirmed in 33.3 % of the patients. CONCLUSIONS: Human metapneumovirus is a major cause of respiratory tract illness in hospitalized infants. This virus causes mainly bronchiolitis and recurrent wheezing and is more frequent in spring. Coinfection with other respiratory viruses is frequent.

[Chest radiograph in bronchiolitis: is it always necessary?]. / Radiografía de tórax en la bronquiolitis: es siempre necesaria?

BACKGROUND: The routine use of chest radiograph in infants with bronchiolitis increases health costs and can often unnecessarily expose the patient to radiation. OBJECTIVES: To evaluate the prevalence of infiltrate/atelectasis in infants younger than 2 years who presented to the emergency department with bronchiolitis, to assess whether patient management is changed after viewing the chest radiograph and to determine which clinical variables can accurately identify children with normal radiographs, with a view to reducing unnecessary radiological investigations. PATIENTS AND METHODS: From October 2003 to December 2004, infants aged < 24 months evaluated in the emergency department of the Severo Ochoa Hospital (Madrid) with a diagnosis of bronchiolitis were included in this study. The variables registered were age, sex, time since onset, respiratory rate, temperature, asymmetry on auscultation, oxygen saturation and the virus identified. A chest radiograph was obtained and the need for admission was evaluated before and after obtaining the results. RESULTS: Two hundred fifty-two infants were included, of which 50 % were aged less than 5 months. Infiltrate/atelectasis was identified in 14.3 % (95 % CI: 10.1-18.5; kappa coefficient: 0.64). Patients with infiltrate/atelectasis were 2.5 times more likely to have a temperature of > or = 38 degrees C (p: 0.004), O2 saturation of < 94 % (p: 0,006) and to be admitted before the results of chest radiograph were known. No differences were found between children with and without infiltrate in age at presentation, sex, disease duration, respiratory rate or identified virus. Patient management was modified in 30 % of patients with infiltrate/ atelectasis. Patients with a temperature of < 38 degrees and O2 saturation of > 94 % had a 92 % probability of normal chest radiograph. CONCLUSIONS: Most infants presenting with bronchiolitis had a normal chest radiograph. Temperature >or = 38 degrees and O2 saturation < 94 % were significantly associated with infiltrate/atelectasis. In most infants with bronchiolitis, the absence of fever and hypoxia are good predictors of normal chest radiographs.

Role of emerging respiratory viruses in children with severe acute wheezing.

BACKGROUND: Acute wheezing episodes are frequently associated with respiratory viral infections in children. However, the role of the recently described respiratory viruses is not yet fully understood. OBJECTIVE: The main objective of this study was to estimate the frequency of human metapneumovirus (HMPV), human bocavirus (HBoV), and 14 other respiratory viruses in hospitalized children with acute wheezing. METHODS: A prospective study was conducted on children <14 years old, admitted with an acute expiratory wheezing episode from September 2005 to June 2008. Viruses were detected in nasopharyngeal aspirates by polymerase chain reaction. Clinical data were prospectively recorded. RESULTS: A viral pathogen was identified in 444 (71%) out of 626 hospitalized acute wheezing episodes. Respiratory syncytial virus (RSV) was the most frequently detected (27%), followed by rhinovirus (24%), adenovirus (17.8%), HBoV (16%), and HMPV (4.7%). The rate of viral detection was significantly higher in infants (77.3%), than in older children (59.8%) (P < 0.001). RSV and HBoV were more prevalent in infants (P < 0.001) than in older children. CONCLUSION: The most prevalent viruses found in severe acute wheezing episodes were RSV and rhinovirus not only in childhood, but also in infancy. However, other emerging viruses such as HBoV and metapneumovirus also play an important role in wheezing episodes.

Human bocavirus infection in a neonatal intensive care unit.

Human bocavirus (HBoV) plays a non-insignificant role as a pathogen in respiratory tract diseases in the pediatric population, especially in infants younger than 2 years of age. In this paper, we have described two cases of a possible nosocomial infection in a neonatal intensive care unit being HBoV the sole detected respiratory virus in clinical samples.

Human metapneumovirus infections in hospitalised infants in Spain.

BACKGROUND: Human metapneumovirus (hMPV) causes lower respiratory tract infections, particularly in young children and the elderly. METHODS: A prospective study was conducted on the clinical characteristics of infants <2 years of age admitted to hospital for respiratory infection and the characteristics of hMPV infections were compared with those of infections caused by respiratory syncytial virus (RSV). Influenza A, B and C viruses, RSV, parainfluenza viruses, and adenoviruses were simultaneously detected in clinical samples by multiple reverse transcription nested-PCR assay. The presence of hMPV was tested in all samples using two separate RT-PCR tests. RESULTS: A respiratory virus was detected in 65.9% of the 749 children included in the study. hMPV, found in 69 of the positive nasopharyngeal aspirates (14%), was the most common virus after RSV. Peak incidence was in March and over 80% of children were <12 months of age. The most common diagnoses were recurrent wheezing (49.3%) and bronchiolitis (46.4%). Oxygen therapy was required by 58% of patients, and assisted ventilation by one. Clinical characteristics in the 18 co-infections were indistinguishable from those of single infections. Fifty one hMPV single infections were compared with 88 hRSV single infections. Recurrent wheezing was diagnosed more frequently in hMPV patients. All other variables tested were similar in both groups. CONCLUSIONS: hMPV was the second most frequent virus after RSV in infants <2 years of age hospitalised for respiratory infection and was associated with lower respiratory tract infections. hMPV occurred predominantly in springtime. Co-infections were frequent and clinically similar to single infections and RSV infections.

Oligonucleotide array for simultaneous detection of respiratory viruses using a reverse-line blot hybridization assay.

The interest in developing new diagnostic methods based on arrays of multiple probes to detect and type simultaneously a wide range of different infectious agents is increasing. This becomes a necessity in the case of infectious agents such as respiratory viruses that cause diseases with very similar signs and symptoms. Such tools will permit rapid and accurate diagnosis of different agents causing respiratory infection leading to the most adequate prevention and/or treatment measures. In this article a reverse-line blot hybridization (RLB) assay for the detection of a wide range of respiratory viruses is presented and evaluated for its usefulness in routine diagnosis. This assay employs an array of 18 oligonucleotide probes immobilized on a nylon membrane. Biotin-labeled PCR products obtained with two multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assays described previously, which allow for the detection of fourteen different groups of respiratory viruses, were hybridized to the oligonucleotide array. Detection was performed using a chemiluminescent method. The standardization of the method showed that the RLB assay could be an alternative to the nested PCR assay for enhancing the sensitivity in the detection of the amplified products, avoiding the problem of cross-over contamination, increasing the specificity, and therefore simplifying the method. This is of main interest in laboratories with few facilities. The feasibility and accuracy of the RT-PCR-RLB assay for detecting respiratory viruses proves that such approach could be a first stage to develop a microarray assay for routine diagnosis of infectious diseases.

Molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region.

Here we present a system for adenovirus detection and genotyping based on PCR amplification and phylogenetic analysis of a conserved hexon gene fragment. The system was validated using 157 sequences (86 previously typed and 71 clinical samples) and correctly identified species and serotype in 100% and 84% of sequences, respectively. Known associations between specific serotypes and clinical syndromes are verified. Possible new associations are described to allow further independent testing.

A new European perspective of influenza pandemic planning with a particular focus on the role of mammalian cell culture vaccines.

Sixteen EU scientists and doctors were interviewed about pandemic planning using psychometric methods applied to a scientific problem for the first time. Criticism was aimed at countries which have no plan whatsoever, the majority of nations. Many such countries have not invested in scientific infrastructure and public health. Amongst the 15 or so published pandemic plans a lack of detail was identified. Of particular need was investment into avian virus vaccine stocks (H1-15), prepared licenses of vaccine and pre purchase and agreed distribution, investment into stocks of antivirals, antibiotics and masks. Most but not all members of the group predicted a global outbreak within 5 years, most probably starting in SE Asia. However it was recognised that a pandemic could start anywhere in the world which had juxtaposition of young people, chickens, ducks and pigs. Mammalian cell culture production using wild type virus with the production factory at category III levels of security was exemplified. Antivirals would be essential to ameliorate the first wave of infection although significant quantities of cell grown vaccine could be produced if, as in 1918, 1957 and 1968 there is a long period between the first virus isolation and person to person spread. The wider scientific community is more energised than previously for very serious preparations to be in place way before the outbreak begins as this is a major public health problem, completely dwarfing concerns about bioterrorism.

Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCR assays.

There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT-nested PCR assay has been used widely for simultaneous detection of non-related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT-PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT-PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT-PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.